The administration of tamoxifen to patients with early stage breast cancer expressing the estrogen receptor (ER) significantly reduces tumor recurrence in many cases, but markers predictive of treatment failure have not been identified. Using gene expression profile analysis of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen only, we identified a novel biomarker that is predictive of tumor recurrence with an overall accuracy of 80%. This biomarker, consisting of a ratio of two genes, the homeobox gene HOXB13 and the interleukin 17B receptor gene, outperforms all existing biomarkers. Furthermore, ectopic expression of one of these genes, HOXB13, in MCF10A breast epithelial cells enhances HER1-and HER2- motility and invasion in vitro, and HOXB13 expression is increased in vivo in preinvasive and invasive primary breast cancers, including a subset of ER-negative tumors. The overall goals of the project are: 1) to validate the clinical utility of this novel two-gene expression biomarker to predict the clinical outcome in early stage breast cancer; and 2) to determine the role of HOXB13 in ER-positive and ER-negative breast cancer. Specifically, in Aims 1 and 2, we propose to validate the predictive utility of this biomarker in a cohort of women with ER positive breast cancer and in a cohort of women with ER-negative breast cancer. We will then, in the second parts of Aims 1 and 2, determine the role of HOXB13 in ER-mediated transcriptional activation and its effects on HER1- and HER2-mediated signaling in in vitro models of ER-positive and ER-negative breast cancer, respectively. Finally, in Aim 3, through genetic crosses between HOXB13 knockout mice and transgenic mice targeting expression of HER2/neu and cyclin D1, we propose to determine whether HOXB13 is necessary for the breast tumorigenesis in mouse models of ER-positive and -negative breast cancer. In Aim 3, we also plan to characterize transgenic mice targeting expression of HOXB13. Accomplishment of these goals will improve identification of patients appropriate for alternative hormonal or chemotherapeutic regimens, and may open new avenues of research into the molecular mechanisms of resistance to tamoxifen.